Kit for assaying EphA4 processing by gamma-secretase

ABSTRACT

The present invention provides a kit for assaying the processing of EphA4 by γ-secretase.

RELATED APPLICATION

This application is a divisional of, and claims priority from, U.S.patent application Ser. No. 12/135,307, filed Jun. 9, 2008 (U.S. Pat.No. 7,910,324), which claims the benefit of the filing date of U.S.Provisional Application Ser. No. 60/942,687 (filed on Jun. 8, 2007; nowabandoned), the entirety of such provisional application is incorporatedby reference herein.

FIELD OF TILE INVENTION

The present invention relates to a screening method using EphA4, whichis a novel substrate for γ-secretase, and a kit for use in the method.

BACKGROUND OF THE INVENTION

γ-Secretase is a complex protein (a member of the aspartate proteasefamily) comprising presenilin, nicastrin, Aph-1 (“anterior pharynxdefective 1”) and Pen-2 (“presenilin enhancer 2”) as basic components.Presenilin is the catalytic domain; the presenilin gene has beenidentified as a causative gene for familial Alzheimer's disease (AD).γ-Secretase acts on single-pass transmembrane proteins as itssubstrates. Representative substrates of γ-secretase are amyloidprecursor protein (APP) and Notch. When cleaved by β-secretase at theβ-site and by γ-secretase at the γ-site, APP produces amyloid βprotein(Aβ). The thus-produced Aβ is classified into peptides with differentlengths depending on the cleavage site in the amino acid sequence(C-terminal side). Of these peptides, Aβ42 which is strongly hydrophobicand ready to aggregate (ready to take the β-sheet structure) exhibitsneurotoxicity. It has been considered that this phenomenon may be themajor cause of Alzheimer's disease. Recently, however, a report has beenmade that presenilin 1 (PS1) and presenilin 2 (PS2) double-knockout micecapable of producing no Aβ show AD-like phenotypes such as decrease ofsynapses and neuronal death; this suggests the existence of a pathogenicmechanism of AD that is independent from APP (see Saura C A, Choi S Y,Beglopoulos V, Malkani S, Zhang D, Shankaranarayana Rao B S, ChattarjiS, Kelleher R J 3rd, Kandel E R, Duff K, Kirkwood A, and Shen J., “Lossof presenilin function causes impairments of memory and synapticplasticity followed by age-dependent neurodegeneration,” Neuron42(1):23-36 (Apr. 8, 2004)).

On the other hand, Eph receptor A4 (EphA4) is a member of the receptortyrosine kinase family, and is a molecule regulating the morphogenesisof post-synapses. It is known that EphA4 knockout or expression of EphA4dominant-negative mutants causes a decrease in the number of dendriticspines (small, thorn-like protrusions found on dendrites) and makes thespine shape slender (see Murai K K, Nguyen L N, Irie F, Yamaguchi Y,Pasquale E B, “Control of hippocampal dendritic spine morphology throughephrin-A3/EphA4 signaling,” Nat Neurosci.; 6(2):153-60 (February 2003)).It is generally proposed that the processes of memory and learning arereflected by the number of and the morphology of the spines.

However, it has never been reported to date that EphA4 is a substratefor γ-secretase.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a screening methodusing EphA4, a novel substrate for γ-secretase, in particular a methodof screening for compounds which affect the processing of EphA4 byγ-secretase.

The present inventors have proved for the first time that EphA4 iscleaved by γ-secretase in HEK293 cells and primary culture ofhippocampal neurons by using a γ-secretase inhibitor(2S)-2-{[(3,5-difluorophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide.The present inventors have also found for the first time that it ispossible to detect the cleavage of EphA4 by using an antibody specificto cleaved EphA4 or an antibody specific to a hemagglutinin tag at theC-terminus of EphA4.

Briefly, the present inventors have demonstrated that a screening methodwith EphA4 utilizing the activity of γ-secretase upon EphA4 (inparticular, cleavage accelerating activity or cleavage inhibitingactivity) is effective by showing that the cleavage of EphA4 isinhibited by γ-secretase inhibitor).

An accelerator for the EphA4 degradation activity of γ-secretase that isobtainable by the screening method of the present invention is acompound which accelerates the processing of EphA4 through γ-secretase.An inhibitor for the EphA4 degradation activity of γ-secretaseobtainable by the screening method of the present invention is acompound which reduces the processing of EphA4 through γ-secretase.According to the present invention, it has become possible to developtherapeutics for memory disorders of interest (preferably AD) byselecting those compounds which act on γ-secretase selectively.

The present invention provides, but is not limited to, the followingembodiments.

In one embodiment, the present invention relates to a method ofscreening for compounds which affect the processing of EphA4 byγ-secretase. More specifically, the method comprises the followingsteps: (a) an assay step of examining the cleavage of EphA4 byγ-secretase, wherein a first biological composition containingγ-secretase or a biologically active fragment thereof is contacted witha second biological composition containing the EphA4 of the presentinvention to thereby measure the cleavage of the EphA4; and (b) a stepof secondarily evaluating whether or not candidate compounds arecompounds which affect γ-secretase, wherein those candidate compoundswhich affect the cleavage of the EphA4 by γ-secretase are selected andthe thus selected compounds are identified as compounds which affect theprocessing of EphA4 by γ-secretase.

In another embodiment, the present invention relates to a screeningmethod further comprising, in addition to the above-described steps, astep of evaluating a candidate compound as a compound which inhibits theprocessing of EphA4 through γ-secretase or an inhibitor for the EphA4degradation activity of γ-secretase, when EphA4 undegraded product inthe presence of the candidate compound was increased relative to EphA4undegraded product in the absence of the candidate compound.

In still another embodiment, the present invention relates to ascreening method further comprising, in addition to the above-describedsteps, a step of evaluating a candidate compound as a compound whichaccelerates the processing of EphA4 through γ-secretase or anaccelerator for the EphA4 degradation activity of γ-secretase, whenEphA4 undegraded product in the presence of the candidate compound wasdecreased relative to EphA4 undegraded product in the absence of thecandidate compound.

In still another embodiment, the present invention relates to ascreening method further comprising, in addition to the above-describedsteps, a step of evaluating a candidate compound as a compound whichaccelerates the processing of EphA4 through γ-secretase or anaccelerator for the EphA4 degradation activity of γ-secretase, whenEphA4 cleavage product in the presence of the candidate compound wasincreased relative to EphA4 cleavage product in the absence of thecandidate compound.

In still another embodiment, the present invention relates to ascreening method further comprising, in addition to the above-describedsteps, a step of evaluating a candidate compound as a compound whichinhibits the processing of EphA4 through γ-secretase or an inhibitor forthe EphA4 degradation activity of γ-secretase, when EphA4 cleavageproduct in the presence of the candidate compound was decreased relativeto EphA4 cleavage product in the absence of the candidate compound.

In still another embodiment, the present invention relates to ascreening method further comprising, in addition to the above-describedmethod, a step of measuring the cleavage of APP or a polypeptidecomprising a γ-secretase cleavage site of APP (hereinafter, expressed as“polypeptide comprising an APP γ-secretase cleavage site”).

In still another embodiment, the present invention relates to ascreening method further comprising, in addition to the above-describedmethod, a step of measuring the cleavage of Notch or a polypeptidecomprising a γ-secretase cleavage site of Notch (hereinafter, expressedas “polypeptide comprising a Notch γ-secretase cleavage site”).

In still another embodiment, the present invention provides apharmaceutical composition comprising at least one compound identifiedby the screening method of the present invention and a pharmacologicallyacceptable carrier. Preferably, the above compound is(2S)-2-{[(3,5-difluorophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide(hereinafter, sometimes referred to as “Compound E”).

In still another embodiment, the present invention provides a method oftreating a disease or condition in which the processing of EphA4 byγ-secretase is abnormal, comprising administering to a patient in needof treatment (preferably a patient with a condition of dementia(preferably AD)) an effective dose of the compound of the presentinvention or a pharmaceutical composition comprising the same(preferably a therapeutic for dementia), wherein preferably the dose iseffective for altering the EphA4 processing activity of γ-secretase inthe cells of the patient.

In still another embodiment, the present invention provides an assay kitfor γ-secretase or a screening kit for identifying γ-secretase inhibitoror accelerator, each of which is applicable to the method of the presentinvention, comprising EphA4, preferably further comprising a substratefor γ-secretase other than EphA4 (preferably APP and/or Notch).

In still another embodiment, the present invention provides a test kitfor measuring the processing of EphA4 by γ-secretase, comprising a firstbiological composition containing γ-secretase or a biologically activefragment thereof and a second biological composition containing EphA4.

According to the present invention, there are provided a method forscreening for compounds which affect the processing of EphA4 byγ-secretase and a kit for use in the method. The compound screened bythe present invention can be a therapeutic for a memory disorder ofinterest, in particular dementia (preferably AD).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the results of analysis of EphA4 processingusing EphA4-transfected 293/EBNA-1 cell strain.

FIG. 2 is a diagram showing the results of analysis of EphA4 processingusing primary culture of rat hippocampal neurons.

DETAILED DESCRIPTION OF THE INVENTION

Herein below, the present invention will be described in more detail.The present specification encompasses the contents disclosed in thespecification and the drawings of U.S. Provisional Patent ApplicationNo. 60/942,687 (filed on Jun. 8, 2007) based on which the present patentapplication claims priority.

The term “patient” as used herein refers to an animal, preferably amammal.

The term “mammal” as used herein means any animal classified as mammal,including human and non-human mammals (such as mouse, rat, hamster,guinea pig, rabbit, pig, dog, horse, cattle, monkey, etc.). Preferably,the mammal in the present specification is human. When the mammal ishuman, the term “patient” include adults and children, and also male andfemale. Children include newborn infants, infants and adolescents.

The term “γ-secretase” as used herein means an enzyme or a complexcomposed of a plurality of molecules, each of which is in charge of theproduction of Aβ by cleaving (degrading) APP within its transmembranedomain. The plurality of molecules comprise at least one moleculeselected from presenilin, nicastrin, Aph-1 and Pen-2. Examples of theγ-secretase of the present invention include mouse Presenilin 1(NM_(—)008943), rat Presenilin1 (D82363), human Presenilin1(NM_(—)000021), mouse Presenilin2 (NM_(—)011183) rat Presenilin2(NM_(—)031087), human Presenilin2 (NM_(—)000447), mouse Nicastrin(NM_(—)021607), rat Nicastrin (NM_(—)174864), human Nicastrin(NM_(—)015331), mouse Aph-1 (NM_(—)146104), rat Aph-1 (NM_(—)001014255),human Aph-1 (NM_(—)016022), mouse Pen-2 (NM_(—)25498), rat Pen-2(NM_(—)001008764) and human Pen-2 (NM_(—)172341). Each componentmolecule of the γ-secretase of the present invention may be afull-length molecule or a part thereof, as long as that γ-secretase hasan enzyme activity equivalent to that of γ-secretase functioning invivo. Further, the γ-secretase of the present invention may be a mutantγ-secretase. The mutant γ-secretase is a polypeptide composed offull-length component molecules which may have deletion, substitution,insertion and/or addition of one or more (preferable one or several)amino acids, or a polypeptide having an amino acid sequence comprising acombination of such mutations, each of the above polypeptide having anenzyme activity equivalent to that of γ-secretase functioning in vivo.

The term “cleavage of EphA4” as used herein refers to an event in whichEphA4, cut by γ-secretase, produces a fragment shorter than the initialEphA4 before cutting. The term “EphA4 undegraded product” used hereinrefers to a polypeptide which is produced as a result of non-cleavage ofEphA4; this polypeptide means the EphA4 polypeptide before degradationby γ-secretase.

The term “biologically active fragment of γ-secretase” as used hereinmeans a fragment having an enzyme activity equivalent to that ofγ-secretase functioning in vivo. Examples of such fragments includefragments capable of cleaving APP or EphA4.

It should be noted that sometimes, the term “γ-secretase” as used hereinis intended to include the “biologically active fragment of γ-secretase”in the present specification.

The term “EphA4” used herein refers to a known polypeptide which is aregulatory factor for synapse formation and/or maintenance (Murai K K etal., Nat Neurosci. 2003 February; 6(2):153-60). The EphA4 of the presentinvention include, but are not limited, to human EphA4 (NM_(—)004438.3and BC026327), rhesus monkey (Macaca mulatta) EphA4 (XM_(—)001106493.1,XM_(—)001106620, XM_(—)001106561, XM_(—)001106876, XM_(—)001106943 andXM_(—)001106806), chimpanzee (Pan troglodytes) EphA4 (XM_(—)001164636.1,XM_(—)001164828, XM_(—)526042, XM_(—)001164899, XM_(—)001164862 andXM_(—)001164676), rat EphA4 (XM_(—)244186.4, SEQ ID NO: 2), mouse EphA4(NM_(—)007936.3, BC052164, AK028509, X65138, BC004782 and AK132203),gray short-tailed opossum (Monodelphis dornestica) EphA4(XM_(—)001365826), dog (Canis familiaris) EphA4 (XM_(—)536084), chicken(Gallus gallus) EphA4 (NM_(—)204781 and Z19059), African clawed frog(Xenopus laevis) EphA4 (NM_(—)001085992, L26099 and NM_(—)001096714) andzebrafish (Danio rerio) EphA4 (NM_(—)001005919 and XM_(—)001342436).Among them, mammal EphA4 polypeptides are preferable. EphA4 comprises aγ-secretase cleavage site, a transmembrane domain and a kinase activitysite in its structure (Aoto, J et al., Brain Res 2006 11). The ligandthereof is the Ephrin A family (Aoto, J et al., Brain Res 2006 11).

In the present invention, EphA4 may be a polypeptide derived from any ofthe above-listed animals, a recombinant polypeptide or a syntheticpolypeptide.

The EphA4 of the present invention may be either a full-lengthpolypeptide or a partial polypeptide thereof, as long as it comprisesthe γ-secretase cleavage site of EphA4.

Further, in the present invention, EphA4 may be a mutant EphA4. Themutant EphA4 is a full-length EphA4 polypeptide which may have deletion,substitution, insertion and/or addition of one or more (preferable oneor several) amino acids, or a polypeptide having an amino acid sequencecomprising a combination of such mutations, each of the abovepolypeptide being functionally substantially identical with EphA4. Thepolypeptide which is “functionally substantially identical with EphA4”means a polypeptide having an activity of EphA4, for example, apolypeptide which has a cleavage activity in a γ-secretase-dependentmanner.

Table 1 below shows correspondence between various animal-derived EphA4polypeptides and their nucleotide or amino acid sequences. Table 2 belowshows correspondence between various animal-derived γ-secretases andtheir nucleotide or amino acid sequences.

TABLE 1 Amino Acid Source or Type Accession No. Nucleotide SequenceSequence Rat XM_244186 SEQ ID NO: 1 SEQ ID NO: 2 (Rattus norvegicus) RatEphA4-HA XM_244186 SEQ ID NO: 3 SEQ ID NO: 4 Human NM_004438 SEQ ID NO:5 SEQ ID NO: 6 (Homo sapiens) BC026327 SEQ ID NO: 7 SEQ ID NO: 8 MouseNM_007936 SEQ ID NO: 9 SEQ ID NO: 10 (Mus musculus) BC052164 SEQ ID NO:11 SEQ ID NO: 12 X65138 SEQ ID NO: 13 SEQ ID NO: 14 BC004782 SEQ ID NO:15 SEQ ID NO: 16 AK132203 SEQ ID NO: 17 SEQ ID NO: 18 Gray short-tailedopossum XM_001365826 SEQ ID NO: 19 SEQ ID NO: 20 (Monodelphis domestica)Dog XM_536084 SEQ ID NO: 21 SEQ ID NO: 22 (Canis lupus familiaris)Chimpanzee XM_001164636 SEQ ID NO: 23 SEQ ID NO: 24 (Pan troglodytes)XM_001164828 SEQ ID NO: 25 SEQ ID NO: 26 XM_526042 SEQ ID NO: 27 SEQ IDNO: 28 XM_001164899 SEQ ID NO: 29 SEQ ID NO: 30 XM_001164862 SEQ ID NO:31 SEQ ID NO: 32 XM_001164676 SEQ ID NO: 33 SEQ ID NO: 34 Rhesus monkeyXM_001106493 SEQ ID NO: 35 SEQ ID NO: 36 (Macaca mulatta) XM_001106620SEQ ID NO: 37 SEQ ID NO: 38 XM_001106561 SEQ ID NO: 39 SEQ ID NO: 40XM_001106876 SEQ ID NO: 41 SEQ ID NO: 42 XM_001106943 SEQ ID NO: 43 SEQID NO: 44 XM_001106806 SEQ ID NO: 45 SEQ ID NO: 46 Chicken NM_204781 SEQID NO: 47 SEQ ID NO: 48 (Gallus gallus) African clawed frog NM_001085992SEQ ID NO: 49 SEQ ID NO: 50 (Xenopus laevis) L26099 SEQ ID NO: 51 SEQ IDNO: 52 NM_001096714 SEQ ID NO: 53 SEQ ID NO: 54 Zebrafish NM_001005919SEQ ID NO: 55 SEQ ID NO: 56 (Danio rerio) XM_001342436 SEQ ID NO: 57 SEQID NO: 58

TABLE 2 Amino Acid Source or Type Accession No. Nucleotide SequenceSequence Mouse Presenilin 1 NM_008943 SEQ ID NO: 59 SEQ ID NO: 60 (Musmusculus) Rat Presenilin 1 D82363 SEQ ID NO: 61 SEQ ID NO: 62 (Rattusnorvegicus) Human Presenilin 1 NM_000021 SEQ ID NO: 63 SEQ ID NO: 64(Homo sapiens) Mouse Presenilin 2 NM_011183 SEQ ID NO: 65 SEQ ID NO: 66(Mus musculus) Rat Presenilin 2 NM_031087 SEQ ID NO: 67 SEQ ID NO: 68(Rattus norvegicus) Human Presenilin 2 NM_000447 SEQ ID NO: 69 SEQ IDNO: 70 (Homo sapiens) Mouse Nicastrin NM_021607 SEQ ID NO: 71 SEQ ID NO:72 (Mus musculus) Rat Nicastrin NM_174864 SEQ ID NO: 73 SEQ ID NO: 74(Rattus norvegicus) Human Nicastrin NM_015331 SEQ ID NO: 75 SEQ ID NO:76 (Homo sapiens) Mouse Aph-1 NM_146104 SEQ ID NO: 77 SEQ ID NO: 78 (Musmusculus) Rat Aph-1 NM_001014255 SEQ ID NO: 79 SEQ ID NO: 80 (Rattusnorvegicus) Human Aph-1 NM_016022 SEQ ID NO: 81 SEQ ID NO: 82 (Homosapiens) Mouse Pen-2 NM_025498 SEQ ID NO: 83 SEQ ID NO: 84 (Musmusculus) Rat Pen-2 NM_001008764 SEQ ID NO: 85 SEQ ID NO: 86 (Rattusnorvegicus) Human Pen-2 NM_172341 SEQ ID NO: 87 SEQ ID NO: 88 (Homosapiens)

The term “substitution” used herein means preferably conservativesubstitution in which one or more (preferably one or several) amino acidresidues are substituted with other chemically similar amino acidresidues so that the activity of the peptide is not substantiallymodified. Examples of conservative substitution include substitution ofa hydrophobic residue with other hydrophobic residue and substitution ofa polar residue with other polar residue with the same electric charge.Functionally similar amino acids which allow such substitution are knownto those skilled in the art for each amino acid. Specifically, examplesof non-polar (hydrophobic) amino acids include alanine, valine,isoleucine, leucine, proline, tryptophan, phenylalanine and methionine;examples of polar (neutral) amino acids include glycine, serine,threonine, tyrosine, glutamine, asparagine and cystein. Examples ofpositively charged (basic) amino acids include arginine, histidine andlysine. Examples of negatively charged (acidic) amino acids includeaspartic acid and glutamic acid.

The number of amino acids which may be deleted, substituted, insertedand/or added as described above is, for example, 1 to 30, preferably 1to 20, more preferably 1 to 10, still more preferably 1 to 5,particularly preferably 1 to 2.

The mutant EphA4 includes those polypeptides which consist of an aminoacid sequence having preferably 80% or more, more preferably 85% ormore, still more preferably 90% or more, still yet more preferably 95%or more, particularly preferably 98% or more, most preferably 99% ormore homology (identity) with the amino acid sequence of SEQ ID NO: 2and have substantially the same activity as that of EphA4 (e.g., anactivity to cause a γ-secretase degradation activity-dependent change).As long as these conditions are satisfied, the mutant EphA4 may be apolypeptide derived from any of the above-listed animals, a recombinantpolypeptide or a synthetic polypeptide, as long as the polypeptide.

The identity described above may be values calculated by homology searchprograms known to those skilled in the art. For example, identity can becalculated by using default parameters in the homology algorithm BLAST(Basic local alignment search tool; http://www.ncbi.nlm.nih.gov/BLAST/)of The National Center for Biotechnology Information (NCBI).

The EphA4 of the present invention includes all EphA4 derivativescomprising at least the γ-secretase cleavage site of EphA4. Thesepolypeptides are particularly useful in detecting and purifying EphA4.

The EphA4 may take any of the following forms: a fusion polypeptidefused to other polypeptide, a tagged or labeled polypeptide or apolypeptide otherwise modified. These polypeptides may be obtained byrecombinant DNA techniques, site-directed mutagenesis, treatment withmutagenic agents (such as hydroxylamine), or automated peptidesynthesis.

Examples of particularly useful systems as tagged EphA4 polypeptidesinclude, but are not limited to, hemagglutinin (HA) system,glutathione-S-transferase (GST) system, maltose-binding protein system,6× histidine system, 8× histidine system and the like.

Examples of modifications incorporating the above-mentioned label orother detectable moieties include, but are not limited to, biotin label,radioactive label, fluorescence label, chemiluminescence label and thelike. The EphA4 of the present invention may be labeled with one, two ormore of these labels.

Examples of antibodies that may be used to monitor the cleavage of EphA4by γ-secretase include, but are not limited to, the following: ananti-EphA4 antibody; an antibody which recognizes EphA4 undegradedproduct produced as a result of non-cleavage of EphA4; or an antibodywhich recognizes EphA4 cleaved product produced as a result of cleavageof EphA4, preferably an antibody which recognizes the intracellulardomain of EphA4, more preferably an antibody which recognizes theC-terminal domain of EphA4. When the undegraded product of a taggedEphA4 polypeptide is to be detected, an antibody which recognizes theselected tag may be used. For example, when an HA tag has been added tothe C-terminus of EphA4, it is possible to detect the undegradation orcleavage of EphA4 using an anti-HA tag antibody. In this case, theantibody is capable of clarifying the presence and concentration of aC-terminal fragment of EphA4 that is produced as a result ofnon-cleavage of EphA4 or the presence and concentration of a C-terminalfragment of EphA4 that is produced as a result of cleavage of EphA4.

In a preferred embodiment of the present invention, EphA4 is a rat EphA4polypeptide, for example, a polypeptide comprising the amino acidsequence as shown in SEQ ID NO: 2. In a still preferred embodiment ofthe present invention, EphA4 is a rat EphA4 polypeptide to which an HAtag is added. For example, a rat EphA4 polypeptide with an HA tag addedat its C-terminus (SEQ ID NO: 4) may be used (Example 1). It is forgranted that polypeptides comprising the entire human EphA4 amino acidsequence or a part thereof (e.g., the amino acid sequence as shown inSEQ ID NO: 6 or 8) may also be used in the same manner as rat EphA4polypeptides.

The present invention further provides a polynucleotide comprising anucleotide sequence encoding the above-described EphA4. One example ofthe polynucleotide encoding the EphA4 of the present invention is apolynucleotide encoding a rat EphA4 (e.g., a polynucleotide as shown inSEQ ID NO: 1). In a preferred embodiment of the present invention, thepolynucleotide encoding EphA4 is a polynucleotide encoding an HA-taggedrat EphA4 polypeptide. For example, a polynucleotide encoding a rat.EphA4 polypeptide with an HA tag added at its C-terminus (SEQ ID NO: 3)may be given (Example 1). It is for granted that polynucleotidescomprising the entire human EphA4 nucleotide sequence or a part thereof(e.g., the nucleotide sequence as shown in SEQ ID NO: 5 or 7) may alsobe used in the same manner as rat EphA4 polynucleotides.

The polynucleotide encoding the EphA4 of the present invention may be apolynucleotide encoding the above-described EphA4 mutant or EphA4derivative. For example, a polynucleotide which comprises a nucleotidesequence having 80% or more, preferably 85% or more, still morepreferably 90% or more, yet still more preferably 95% or more,particularly preferably 98% or more, most preferably 99% or morehomology (identity) with the nucleotide sequence as shown in SEQ ID NO:1 and encodes a polypeptide having substantially the same activity asthat of EphA4 may be included.

Further, the polynucleotide encoding the EphA4 of the present inventionincludes a polynucleotide which hybridizes to a polynucleotideconsisting of a nucleotide sequence complementary to the nucleotidesequence as shown in SEQ ID NO: 1 under stringent conditions and yetencodes a polypeptide having substantially the same activity as that ofEphA4. The stringent conditions refer to, for example, “2×SSC, 0.1% SDS,42° C.” or “1×SSC, 0.1% SDS, 37° C.” as washing conditions afterhybridization. As more stringent conditions, “1×SSC, 0.1% SDS, 65° C.”or “0.5×SSC, 0.1% SDS, 50° C.” may be given, for example. Morespecifically, a method using Rapid-hyb buffer (Amersham Life Science)may be employed in which pre-hybridization is performed at 68° C. formore than 30 minutes; then, with addition of a probe, a hybrid is formedwhile keeping the reaction solution at 68° C. for more than 1 hour,followed by washing in 2×SSC, 0.1% SDS at room temperature for 20minutes 3 times, washing in 1×SSC, 0.1% SDS at 37° C. for 20 minutes 3times and finally washing in 1×SSC, 0.1% SDS at 50° C. for 20 minutestwice. Alternatively, other method may be employed in whichpre-hybridization is performed in Expresshyb Hybridization Solution(CLONTECH) at 55° C. for more than 30 minutes; then, with addition of alabeled probe, the reaction solution is incubated at 37-55° C. for morethan 1 hour, followed by washing in 2×SSC, 0.1% SDS at room temperaturefor 20 minutes 3 times, washing in 1×SSC, 0.1% SDS at 37° C. for 20minutes once. In these methods, more stringent conditions may beachieved, for example, by raising the temperature of prehybridization,hybridization or the second washing. For example, the temperatures ofprehybridization and hybridization may be raised to 60° C.,respectively; for more stringent conditions, the temperatures may beraised to 68° C., respectively. Those skilled in the art couldappropriately select conditions for obtaining EphA4 isoforms, allelicmutants, and corresponding genes derived from other organisms, by takinginto account various conditions such as probe concentration, probelength, reaction time, etc. in addition to the above-described saltconcentrations and reaction temperatures.

Such polynucleotides may be obtained by gene amplification techniques,hybridization techniques, recombinant DNA techniques, and the like.

The term “biological composition” used herein means a compositioncomprising γ-secretase or a biologically active fragment thereof, orEphA4, and is not particularly limited. For example, the biologicalcomposition may be a cell-free reconstruction system, a mammal or a partthereof, or a transgenic non-human mammal so engineered to overexpressAPP or a part of this transgenic mammal.

In the expressions “first biological composition containing γ-secretaseor a biologically active fragment thereof” and “second biologicalcomposition containing EphA4” used herein, the γ-secretase and/or EphA4may be either endogenous or exogenous.

When the γ-secretase or EphA4 is endogenous, the composition may be anycomposition as long as it contains γ-secretase or EphA4 derived from theabove-mentioned animal or a part thereof.

The term “a part of an animal” include tissues, cells, cell lysates,cell membrane fractions or purified membranes of the above-mentionedanimal. As examples of the cells, cells in the central nervous system;neurons such as brain-derived neurons, cerebral cortex-derived neurons,cerebral cortex-derived primarily cultured neurons, orhippocampus-derived primarily cultured neurons; or glial cells may beenumerated. The γ-secretase or EphA4 may be in the state of beingcontained in a mammal or a part thereof. Alternatively, the γ-secretaseor EphA4 may be a γ-secretase fraction or an EphA4 fraction of celllysate prepared from a mammal. The cell lysate may be obtained bysubjecting γ-secretase- or EphA4-containing cells to lysis with ahypotonic solution or surfactant, or to sonication or other physicaldisruption. Optionally, the cell lysate may be purified with somepurification means such as columns.

When the γ-secretase or EphA4 is exogenous, the biological compositionmay be γ-secretase expressing cells or EphA4 expressing cells preparedby allowing host cells to express the whole or a part of the sequencesin expression vectors comprising a polynucleotide encoding theindividual molecules constituting γ-secretase or a polynucleotideencoding EphA4. Alternatively, the biological composition may be theγ-secretase fraction of a cell lysate derived from γ-secretaseexpressing cells, or the EphA4 fraction or cell membrane fraction of acell lysate derived from EphA4 expressing cells. The cell lysate may beobtained by subjecting γ-secretase- or EphA4-containing cells to lysiswith a hypotonic solution or surfactant, or to sonication or physicaldisruption. Optionally, the cell lysate may be purified with somepurification means such as columns. The expression vector may be avector which is transformed or transfected into a host cell andtemporarily expresses the gene of interest. Alternatively, theexpression vector may be a vector which is integrated into the genome ofa host cell and expresses the gene of interest stably.

The term “transformation” or “transfection” used herein means any andall methods which change DNA contents in eukaryotic cells ormicroorganisms. These methods include calcium phosphate transfection,protoplast fusion transfection, electroporation transfection,DEAE-dextran transfection, liposome transfection, polybrene transfectionand direct microinjection transfection (Sambrook et al., MolecularCloning 3: 16.30-16.31 (1989)).

The host cell into which the above-described expression vector is to betransformed or transfected may be any cell (or cell line) ormicroorganism capable of expressing the gene of interest. Known culturedcells may be used as host cells. Examples of mammal cells or cell lineswhich may be used as host cells include HEK 293 cells, Chinese hamsterovary (CHO) cells, fibroblast cells, primary endothelial cells (HUVECcells), human glioma cells, HeLa cells, COS cells, PC12 cells,lymphoblast cells, melanoma cells, hybridoma cells, oocytes andembryonic stem cells. Examples of known microorganisms which may be usedas host cells include Escherichia coli and yeast. Insect cells such asBmN4 cells may also be used.

The expression vector used in the above-described transformation ortransfection is not particularly limited as long as the vector comprisesa polynucleotide encoding the individual molecules constitutingγ-secretase or a polynucleotide encoding EphA4. Such an expressionvector may be a plasmid obtainable by introducing the polynucleotideinto a known expression vector selected appropriately depending on thehost cell to be used.

Examples of the above-mentioned known expression vector include pUC,pTV, pGEX, pKK or pTrcHis for E. coli; pEMBLY or pYES2 for yeast;pcDNA3, pMAMneo and pBabe Puro for CHO cells, HEK293 cells or COS cells;and a vector comprising the polyhedrin promoter of Bombyx mori nuclearpolyhedrosis virus (BmNPV) (such as pBD283) for BmN4 cells.

Since a promoter functions effectively for giving a strong transcriptionactivity in mammal cells, the expression plasmid preferably comprises apromoter. Examples of promoters include CMV immediate early promoter,retrovirus promoters (e.g., LTR from MLV or MMTV), promoters of SV40,RSV LTR, HIV-1 LTR and HIV-2 LTR, adenovirus promoters (e.g., those fromE1A region, E2A region or MLP region), and promoters of AAVLTR,cauliflower mosaic virus, HSV-TK and avian sarcoma virus.

The above-described transformed or transfected host cell is notparticularly limited as long as the host cell comprises a polynucleotideencoding the individual molecules constituting γ-secretase or apolynucleotide encoding EphA4. For example, the transformed cell may bea transformant in which the polynucleotide has been integrated into thechromosome thereof. Alternatively, the transformed cell may be atransformant comprising the polynucleotide in the form of a plasmid. Itis also possible that the transformed cell is a transformant which isnot expressing γ-secretase or EphA4. These transformants may be obtainedby transforming a desired host cell with the above-mentioned plasmid orthe above-described polynucleotide per se.

A cell containing the above-described γ-secretase and/or EphA4 is notparticularly limited as long as the cell is capable of expressingγ-secretase and/or EphA4 on the surface of its cell membrane. Asexamples of such cells, a cell expressing endogenous γ-secretase andendogenous EphA4, a cell expressing γ-secretase and EphA4 one of whichis endogenous and the other is exogenous or a cell expressing exogenousγ-secretase and exogenous EphA4 may be given. Such cells may also beobtained by culturing under conditions which allow expression ofγ-secretase and/or EphA4. Alternatively, such cells may be obtained byinjecting into an appropriate cell an RNA encoding the individualmolecules constituting γ-secretase and/or an RNA encoding EphA4 andculturing the resultant cell under conditions which allow expression ofγ-secretase and/or EphA4.

The above-described cell membrane fraction may be obtained, for example,by disrupting cells expressing the γ-secretase or EphA4 of the presentinvention and isolating cell membrane-rich fractions. As methods fordisrupting cells, homogenizing in a homogenizer; disrupting in a Waringblender or Polytron; disrupting by sonication; ejecting cells from athin nozzle while applying pressure with a French press; and so on maybe used. As methods for fractionating cell membrane, fractionationmethods with centrifugal force such as differential centrifugation ordensity gradient centrifugation may be used.

For purification, a known method for protein purification may be used.The method comprises a step of crudely fractionating cells intopolypeptide fractions and non-polypeptide fractions. After theγ-secretase or EphA4 of the present invention has been isolated fromother polypeptides with a column or the like, the desired γ-secretase orEphA4 is further purified by chromatography or electrophoresis tothereby achieve partial purification or complete purification (orhomogeneity by purification). Examples of analysis methods particularlysuitable for preparation/purification of pure peptides includeprecipitation using ammonium sulfate, PEG antibodies, etc.;centrifugation after thermal denaturation; chromatography step (e.g.,ion exchange chromatography, gel filtration chromatography,reversed-phase chromatography, hydroxyapatite chromatography, affinitychromatography, fast protein liquid chromatography (FPLC), highperformance liquid chromatography (HPLC) or immobilized metal ionaffinity chromatography (IMAC)); isoelectric focusing; gelelectrophoresis; SDS (sodium dodecyl sulfate)-polyacrylamideelectrophoresis (SDS-PAGE); and a combination of these methods and othermethod. Alternatively, γ-secretase or EphA4 may be tagged in advance;then, a crude polypeptide may be applied to a purification column towhich a protein that recognizes the tag is bound; the desiredγ-secretase or EphA4 adsorbed onto the column may be desorbed from thecolumn by feeding an appropriate solvent thereinto. Various purificationsteps may be performed in a different order, or some of the steps may beomitted. A preferable method for evaluating the purity of a fraction isa method in which the specific activity of the fraction is calculatedand compared with the specific activity of the first extract, followedby calculation of the magnitude of purity for evaluation.

The term “APP” used herein β-amyloid precursor protein (βAPP) or amutant thereof. APP is a single-pass transmembrane protein comprising anAβ domain in its C-terminal region, expressed in a large variety ofcells in many mammals. In human, APP is encoded by the gene APP locatedin the long arm of chromosome No. 21 and has three major isotypes(APP695, APP751 and APP770). APP695, APP751 and APP770 consist of 695,751 and 770 amino acid residues, respectively. Examples of APP proteinsinclude human APP695 (NM_(—)201414, NP_(—)958817 and P05067-4), humanAPP751 (NM_(—)201413, NP_(—)958816 and P05067-8), human APP770(NM_(—)000484, NP_(—)000475, P05067-1 and P05067), mouse APP695(NM_(—)007471, NP_(—)031497 and P12023-2), mouse APP751 (P12023-3),mouse APP770 (AY267348, AAP23169, P12023-1 and P12023), rat APP695(P08592-2), rat APP751 (P08592-7) and rat APP770 (NM_(—)019288,NP_(—)062161, P08592-1 and P08592). Examples of APP mutants includeSwedish FAD double mutant, London mutant, valine717 to phenylalaninemutant, valine717 to isoleucine mutant and valine717 to glycine mutant.

The term “Aβ” used herein means the term (β-amyloid protein, amyloid βprotein, β-amyloid peptide, amyloid β peptide or amyloid beta. Forexample, Aβ is a peptide consisting of about 33-40 amino acids residuesin human APP695 amino acid isotype. Preferably, Aβ includes any peptidecomprising a part or all of the amino acid residues from positions 597to 640 in APP, and means every peptide produced from APP by itsN-terminal protein degradation and subsequent C-terminal proteindegradation. Aβ40 and Aβ42 are peptides comprising 40 amino acidresidues and 42 amino acid residues, respectively.

The term “Notch” used herein refers to one of the competing substratesfor γ-secretase belonging to the cell surface receptor Notch family. Forexample, human Notch 1 (AF308602.1), mouse Notch 1 (NM_(—)008714.2) andrat Notch 1 (NM_(—)001105721.1) may be enumerated. Since Notch 1 has animportant function in hematopoiesis, inhibition of the processing ofNotch 1 may potentially cause immunodeficiency and anemia.

The term “candidate compound” used herein means a compound which istested in the compound screening method. Although every molecule may beused as a candidate compound, preferably a compound capable of changingthe activity of γ-secretase (preferably the activity of γ-secretase ofmammals) is used. The candidate compound is one or more compoundscontained in expression products of gene libraries; natural or syntheticlow molecular weight compound libraries; nucleic acids (oligo DNA oroligo RNA); natural or synthetic peptide libraries; antibodies;substances released from bacteria (including those released frombacteria as a result of metabolism); cell (microorganism, plant cell oranimal cell) extracts; cell (microorganism, plant cell or animal cell)culture supernatants; purified or partially purified peptides; extractsfrom marine organisms, plants or animals; soils; or random phage peptidedisplay libraries. The above-described compound may be either a novelcompound or a known compound. Further, the above-described compound maybe a compound modified by conventional chemical means, physical meansand/or biochemical means. For example, the above-described compound maybe a structural analogue which is obtained by subjecting the initialcompound to direct chemical modification (such as acylation, alkylation,esterification or amidation) or random chemical modification. Thecandidate compound may also be a compound identified by pharmacophoresearch of the above-described compound or structure comparison programswith computer. The candidate compound may be in the form of a salt.Further, the candidate compound or a salt thereof may be in the form ofa solvate (including hydrate).

Further, the candidate compound may be a known γ-secretase acceleratoror γ-secretase inhibitor involved in the processing of APP and/or theprocessing of Notch, or a structural analogue of the above acceleratoror inhibitor. A known compound which accelerates or inhibits theactivity of γ-secretase and/or the processing of APP and/or theprocessing of Notch may be a compound that can be designed throughrational drug design. For example, DAPT(N—[N-(3,5-difluorophenacetyl)-L-alanyl]S-phenylglycine t-butyl ester),CM256 and(2S)-2-{[(3,5-difluorophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide(Alexis Biochemicals) may be enumerated.

The expression “compound which affects the processing of EphA4 byγ-secretase” as used herein means either a compound which inhibits theEphA4 cleavage activity by γ-secretase (γ-secretase inhibitor) or acompound which accelerates the EphA4 cleavage activity by γ-secretase(γ-secretase accelerator). It should be noted that γ-secretase inhibitorincludes antagonist and that γ-secretase accelerator includes agonist,γ-Secretase inhibitor and γ-secretase accelerator also include thosecompounds which alter the cleavage site of EphA4 cleavage products byγ-secretase to thereby produce EphA4 cleavage products with differentpeptide lengths.

The term “salt” as used herein refers to a pharmacologically acceptablesalt, and is not particularly limited as long as it forms apharmacologically acceptable salt with the above-described compound.Preferred examples thereof are hydrohalogenic acid salts (such ashydrofluoride, hydrochloride, hydrobromide or hydroiodide), inorganicacid salts (such as sulfate, nitrate, perchlorate, phosphate, carbonateor hydrogencarbonate), organic carboxylic acid salts (such as acetate,oxalate, maleate, tartrate, fumarate or citrate), organic sulfonic acidsalts (such as methanesulfonate, trifluoromethanesulfonate,ethanesulfonate, benzenesulfonate, toluenesulfonate orcamphorsulfonate), amino acid salts (such as aspartate or glutamate),quaternary amine salts, alkali metal salts (such as lithium salt, sodiumsalt or potassium salt) and alkaline earth metal salts (such asmagnesium salt or calcium salt).

According to the first embodiment of the present invention, there areprovided (a) an assay method for examining the cleavage of EphA4 and (b)a method of secondarily evaluating whether or not a candidate compoundis a compound which affects γ-secretase (screening method) utilizing theassay method. As a method of the present invention, the method asdescribed below is provided. The method of the present invention ischaracterized by the use of EphA4, a novel substrate for γ-secretase.The method of the present invention may be performed in an in vitroappropriate cell line or a cell-free system.

The first embodiment of the present invention is capable of evaluatingthe cleavage of EphA4 by γ-secretase after incubating EphA4 andγ-secretase in the presence and absence of a candidate compound.

The method of screening for compounds which affect the processing ofEphA4 by γ-secretase (which is the first embodiment of the presentinvention) comprises the following steps:

-   (i) contacting a first biological composition containing γ-secretase    or a biologically active fragment thereof with a second biological    composition containing EphA4 in the presence of a candidate compound    or EphA4 in the absence of a candidate compound-   (ii) measuring the cleavage of the EphA4 in the presence and absence    of the candidate compound;-   (iii) selecting those candidate compounds which affect the cleavage    of the EphA4 by γ-secretase; and-   (iv) identifying the candidate compounds selected in step (iii) as    compounds which affect the processing of EphA4 by γ-secretase.

The method according to this embodiment may be performed in anappropriate cell line containing γ-secretase and EphA4 or a cell-freesystem containing γ-secretase and EphA4.

The cell line containing γ-secretase and EphA4 may be either a cell lineexpressing endogenous genes or a cell line containing an exogenousgene(s). It is possible to contact a first biological compositioncontaining γ-secretase with a second biological composition containingEphA4 by culturing a cell containing γ-secretase and EphA4 in anappropriate medium in the presence and absence of a candidate compoundand incubating the cell under reaction conditions which allow thecleavage of EphA4 by γ-secretase activity. If a cell line containing anexogenous gene(s) is used, the above-described contact may be performedunder culture conditions which allow the expression of the exogenousgene. It is also possible to apply conditions that allow the cleavage ofother γ-secretase substrate, e.g., reaction conditions known to thoseskilled in the art when the substrate is APP.

Examples of reaction conditions are enumerated below. For cell linesexpressing endogenous genes, in case of primary culture of neurons,culture conditions are in MEM (Invitrogen) medium supplemented with 5%FBS (Hyclone), 1× B27 Supplement (Invitrogen), 0.5 mM L-glutamine(Invitrogen), 25 μg/ml insulin (SIGMA) and 8 μM AraC (SIGMA); under 5%CO₂; and at 37° C. For cell lines containing an exogenous gene(s), inthe case of HEK293 cell line, culture conditions are in 10% FBS(Hyclone)/DMEM (Invitrogen), under 5% CO₂ and at 37° C.

In cell-free systems, a first biological composition containingγ-secretase or a biologically active fragment thereof (e.g., cellmembrane fraction containing γ-secretase) and a second biologicalcomposition containing EphA4 (e.g., cell membrane fraction containingEphA4) may be contacted with each other by incubating these compositionsby mixing them in the presence and absence of a candidate compound.These compositions may be mixed under reaction conditions which allowthe cleavage of EphA4 by γ-secretase activity, e.g., 10 mM HEPES, pH7.4, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM 1,10-phenanthroline, 10μg/ml phosphoramidon, Complete protease inhibitor cocktail (RocheBiochemicals) (Tomita et al., Molecular Neurodegeneration 2006 1:2).Alternatively, these compositions may be mixed under conditions whichallow the cleavage of other γ-secretase substrate, e.g., reactionconditions known to those skilled in the art when the substrate is APP.γ-Secretase or EphA4 may be a purified γ-secretase or EphA4, abiologically active fragment of γ-secretase or EphA4, an analogue ofγ-secretase or EphA4, or a mutant of γ-secretase or EphA4.

When a first biological composition containing γ-secretase is contactedwith a second biological composition containing EphA4 as describedabove, EphA4 cleavage reaction by γ-secretase occurs.

The candidate compound may be added generally within a range fromapprox. 1 nM to 1 mM, usually within a range from approx. 10 μM to 1 mM.In order to identify a compound which changes the cleavage of EphA4 byγ-secretase, the steps described above are performed in the presence andabsence of a candidate compound. Then, the EphA4 cleavage activity ofγ-secretase in the presence of the candidate compound is compared withthat activity in the absence of the candidate compound to evaluate theability of the candidate compound. Thus, a compound which changes thecleavage of EphA4 by γ-secretase is identified. Even a slight change inthe quantity or degree of EphA4 in the presence of a candidate compoundindicates that the EphA4 cleavage activity of γ-secretase has beenchanged in the presence of the candidate compound. Therefore, thecandidate compound can be identified as a compound which affects theprocessing of EphA4 by γ-secretase. For example, a compound whichincreases EphA4 cleavage product or decreases EphA4 undegraded productcompared with control is evaluated as an accelerator for the EphA4degradation activity of γ-secretase. On the other hand, a compound whichdecreases EphA4 cleavage product or increases EphA4 undegraded productcompared with control is evaluated as an inhibitor for the EphA4degradation activity of γ-secretase. The accelerator for EphA4degradation activity obtained by the method of the present invention ispotentially useful for treatment of AD.

When EphA4 is tagged, it is possible to detect the undegraded product orcleavage product of EphA4 using a substance which binds to the tag(e.g., antibody). For example, EphA4 with a hemagglutinin tag added toits C-terminus may be detected using a known anti-HA antibody.

Analysis of EphA4 cleavage is performed by measuring an indicator ofcleavage for one or both of the N-terminal fragment and C-terminalfragment of EphA4.

For analyzing the cleavage of EphA4 by γ-secretase, the followingantibodies may be used: anti-EphA4 antibodies; antibodies recognizing anEphA4 derivative (EphA4 undegraded product) before degradation byγ-secretase, produced as a result of non-cleavage of EphA4; antibodiesrecognizing an EphA4 cleavage product produced as a result of cleavageof EphA4; or antibodies recognizing the intracellular domain of EphA4.

When an undegraded product of tagged EphA4 and/or a cleavage product oftagged EphA4 is to be detected, antibodies to the selected tag may beused.

For example, when an undegraded product of EphA4 polypeptide is to bedetected, an HA tag may be added to the C-terminus of EphA4 polypeptide,followed by detection of the undegraded product with an anti-HA tagantibody. In this case, it is possible to clarify the presence andconcentration of a C-terminal fragment of EphA4 produced as EphA4undegraded product, by detecting and quantitatively determining the HAtag. When EphA4 or tagged EphA4 is labeled, detection may be performedby detecting or quantitatively determining the label.

On the other hand, when a cleavage product of EphA4 polypeptide is to bedetected, the membrane fraction is purified from EphA4-expressing cells;then, the purified membrane fraction is subjected to cleavage reactionby γ-secretase to thereby allow the EphA4 cleavage product to bereleased from the membrane fraction. When this reaction product iscentrifuged, the EphA4 undegraded product is precipitated into themembrane fraction. Thus, the membrane fraction and other fractions canbe separated. By detecting the released fragment, the EphA4 cleavageproduct can be detected. For this detection, an antibody recognizing theintracellular domain of EphA4 is used when endogenous EphA4 is used.When exogenous recombinant EphA4 is used, a cDNA encoding EphA4 with atag sequence added to its C-terminus is used to express the recombinantEphA4. Then, the EphA4 cleavage product is detected using an antibodythat recognizes the tag. The tag is not particularly limited. Forexample, HA, Myc, FLAG or the like may be used.

The antibody to EphA4 is not particularly limited as long as theantibody recognizes EphA4 and is preferably an antibody which recognizesthe intracellular domain of EphA4. For example, the antibody describedin Tremblay et al., J. Comp. Neurol 501 691-702 or a commercial anti-ratEphA4 antibody (Upstate, Zymed or Santacruze) may be used.

Those skilled in the art could prepare an antibody which recognizesEphA4 by immunizing an animal with an immunogen (antigen) and followingthe conventional, general procedures for preparing monoclonalantibodies. As the immunogen, for example, EphA4 or a fragment thereof,or a fusion protein prepared by adding a tag or label to EphA4 or afragment thereof may be used.

For example, a non-human mammal is immunized with the immunogen aloneor, if necessary, together with Freund's adjuvant. Polyclonal antibodiesmay be obtained from the serum collected from the immunized animal.Monoclonal antibodies may be obtained by fusing antibody producing cellsfrom the immunized animal with myeloma cells without autoantibodyproducing ability to prepare fusion cells (hybridomas), cloning thehybridomas, and selecting those clones which produce a monoclonalantibody showing specific affinity to the antigen used for immunizingthe animal. The preparation of monoclonal antibodies from hybridomas maybe performed in vitro. Alternatively, the preparation may be performedin vivo in a non-human mammal, preferably mouse or rat, more preferablyin the abdominal dropsy in mouse, Monoclonal antibodies may be isolatedfrom the resultant culture supernatant or the abdominal dropsy of themammal. The isolation and purification of monoclonal antibodies may beperformed by subjecting the above-mentioned culture supernatant orabdominal dropsy to methods such as saturated ammonium sulfateprecipitation, euglobulin precipitation, caproic acid method, caprilicacid method, ion exchange chromatography (DEAE, DE52, etc.), or affinitycolumn chromatography using anti-immunoglobulin column or protein Acolumn. The monoclonal antibody include those monoclonal antibodiesconsisting of heavy chains and/or light chains having the amino acidsequences which have deletion, substitution or addition of one orseveral amino acids in the heavy chains and/or light chains constitutingthe initial antibody.

In another embodiment of the present invention, it is possible toevaluate whether or not a candidate compound affects the processing ofAPP and/or Notch, in parallel with, simultaneously with, or before orafter the above-described first embodiment of the present invention. Forexample, a step of measuring the cleavage of APP or a polypeptidecontaining an APP cleavage site by γ-secretase (i.e., polypeptidecontaining APP γ-secretase cleavage site) may be included in parallelwith, simultaneously with, or before or after the above-described methodof the present invention. Further, a step of measuring the cleavage ofNotch or a polypeptide containing a Notch cleavage site by γ-secretase(i.e., polypeptide containing Notch γ-secretase cleavage site) may beincluded.

By including such steps, it is possible to evaluate whether or not acandidate compound selectively acts on the processing of EphA4 comparedto the processing of APP and/or Notch. The expression “selectively acton” used herein means to have more inhibitory effect or acceleratingeffect upon the cleavage of substrate EphA4 by γ-secretase than upon thecleavage of other substrate(s). Specifically, according to thisembodiment of the present invention, it is possible to identify acompound which selectively acts only on the processing of EphA4; acompound which selectively acts only on the processing of APP; acompound which selectively acts only on the processing of Notch; or acompound which selectively acts on the processing of APP and EphA4.

As a candidate compound in drug development, a compound which acts inthe same manner as metabolic activity in healthy animals in vivo or acompound which regulates the metabolic activity is preferable. Apreferable example of such a candidate compound is a compound whichinhibits the production of Aβ42 by inhibiting the APP cleavage activityof γ-secretase, does not inhibit the EphA4 cleavage activity ofγ-secretase, and does not inhibit the Notch cleavage activity ofγ-secretase. Another preferable example is a compound which inhibits theproduction of Aβ42 by accelerating the production of Aβ40 throughacceleration of the APP cleavage activity of γ-secretase; acceleratesthe degradation of EphA4 by accelerating the EphA4 cleavage activity ofγ-secretase; and does not inhibit the Notch cleavage activity ofγ-secretase.

As methods for measuring the cleavage by γ-secretase of APP, Notch or apolypeptide containing an APP or Notch γ-secretase cleavage site, assaymethods known to those skilled in the art may be applicable (Song et al.PNAS 96:6959-6963 (1999); Moehlmann et al. PNAS 99:8025-8030 (2002)).For example, a first biological composition containing γ-secretase or abiologically active fragment thereof may be contacted with a biologicalcomposition containing APP or a polypeptide containing an APPγ-secretase cleavage site or a biological composition containing Notchor a polypeptide containing a Notch γ-secretase cleavage site in thepresence and absence of a candidate compound, and then may be measuredthe cleavage of the APP or the polypeptide containing an APP γ-secretasecleavage site or the cleavage of the Notch or the polypeptide containinga Notch γ-secretase cleavage site. This measurement may be performed bymeasuring the cleavage product from the APP or the polypeptidecontaining an APP γ-secretase cleavage site or the cleavage product fromthe Notch or the polypeptide containing a Notch γ-secretase cleavagesite. As one example of the cleavage product from APP or a polypeptidecontaining an APP γ-secretase cleavage site, Aβ may be given. Thequantity of Aβ may be measured, and changes in the quantity between thepresence and absence of the candidate compound may be compared.Alternatively, the degree of cleavage and the quantity of cleavageproduct may be measured by using a known antibody which recognizes thecleavage product from APP or a polypeptide containing an APP γ-secretasecleavage site or the cleavage product from Notch or a polypeptidecontaining a Notch γ-secretase cleavage site. As the antibody whichrecognizes the cleavage product from APP or a polypeptide containing anAPP γ-secretase cleavage site, commercial antibodies (Sigma or Chemicon)may be used. The measurement may be performed, for example, by Westernblotting. As the antibody which recognizes the cleavage product fromNotch or a polypeptide containing a Notch γ-secretase cleavage site,commercial antibodies (Santa Cruz Biotechnology) may be used. Themeasurement may be performed, for example, by Western blotting.

The method of the present invention also includes a high through putscreening (HTS) known to those skilled in the art, which tests a largenumber of compounds simultaneously (see U.S. Pat. No. 5,876,946; U.S.Pat. No. 5,902,732; Jayawickreme and Kost, Curr. Opin. Biotechnol.8:629-634 (1997); Houston and Banks, Curr. Opin. Biotechnol. 8:734-740(1997)).

The method of the present invention also includes the use of known modelanimals. It is possible to analyze the in vivo effect of a compoundselected by the in vitro method of the present invention by using, forexample, an APP processing and/or AD non-human model. APP transgenicnon-human animal models are well-known in the art. One example thereofis Tg2576 mouse (J. Neurosci. 21(2):372-381 (2001); J. Clin. Invest.112:440-449 (2003)). For example, the following analyses may be madeafter administering to Tg2576 mouse a known γ-secretase inhibitor DAPT,(2S)-2-{[(3,5-difluorophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide(Alexis Biochemicals) or a compound of the present invention: evaluationby a method of measuring the Aβ quantities in the brain, cerebrospinalfluid and serum of the mouse (J. Pharmacol. Exp. Ther. 305:864-871(2003); pathological examination of changes in the brain (e.g., changesin Aβ yield, the degree of cerebral atrophy, etc.) resulted from changesin γ-secretase activity; and evaluation of the survival ratio, momentumor food consumption of the mouse.

The pharmaceutical composition comprising the compound identified by themethod of the present invention, preferably the AD therapeutic of thepresent invention, may be administered to patients in various formsthrough an oral or parenteral (e.g., intravenous injection, muscleinjection, subcutaneous administration, rectal administration ortransdermal administration) route. Therefore, the pharmaceuticalcomposition comprising the compound of the present invention may beformulated into various preparations using a pharmacologicallyacceptable carrier by a conventional method depending on theadministration route, though the pharmaceutical composition may be usedalone.

Preferred dosage forms include oral preparations such as tablets,powders, subtle granules, granules, coated tablets, capsules, syrups andtroches; and parenteral preparations such as inhalants, suppositories,injections (including drops), ointments, eye drops, ophthalmicointments, nasal drops, ear drops, cataplasms, and lotions andliposomes.

Examples of carriers used in the formulation include conventionally usedfillers, binders, disintegrants, lubricants, coloring agents andflavoring agents, as well as stabilizers, emulsifiers, absorbefacients,surfactants, pH adjusting agents, antiseptics, antioxidants, expanders,wetting agents, surface activators, dispersing agents, buffers,preservatives, dissolution aids and analgesic agents according tonecessity. They can be formulated according to a conventional procedureusing components commonly used as raw materials for pharmaceuticalpreparations. Examples of nontoxic these components which may be used inthe present invention include animal and vegetable oils such as soybeanoil, beef tallow and synthetic glycerides; hydrocarbons such as liquidparaffins, squalane and solid paraffins; ester oils such as octyldodecylmyristate and isopropyl myristate; higher alcohols such as cetostearylalcohol and behenyl alcohol; silicone resins; silicone oils; surfactantssuch as polyoxyethylene fatty acid esters, sorbitan fatty acid esters,glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters,polyoxyethylene hydrogenated castor oils andpolyoxyethylene-polyoxypropylene block copolymers; water-solublepolymers such as hydroxyethyl cellulose, polyacrylic acids, carboxyvinylpolymers, polyethylene glycol, polyvinylpyrrolidone and methylcellulose;lower alcohols such as ethanol and isopropanol; polyhydric alcohols(polyols) such as glycerol, propylene glycol, dipropylene glycol,sorbitol and polyethylene glycol; sugars such as glucose and sucrose;inorganic powders such as silicic anhydride, magnesium aluminiumsilicate and aluminium silicate; inorganic salts such as sodium chlorideand sodium phosphate; and purified water.

The fillers include, for example, lactose, fructose, corn starch, whitesugar, glucose, mannitol, sorbitol, crystalline cellulose and silicondioxide. The binders include, for example, polyvinyl alcohol, polyvinylether, methylcellulose, ethylcellulose, gum arabic, gum tragacanth,gelatin, shellac, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polypropylene glycol-polyoxyethyleneblock polymers and meglumine. The disintegrants include, for example,starch, agar, gelatin powder, crystalline cellulose, calcium carbonate,sodium hydrogencarbonate, calcium citrate, dextrin, pectin andcarboxymethylcellulose calcium. The lubricants include, for example,magnesium stearate, talc, polyethylene glycol, silica and hardenedvegetable oils. The coloring agents may be any coloring agents which areapproved to be added to pharmaceutical preparations. The flavoringagents include, for example, cocoa powder, menthol, aromatic powder,peppermint oil, camphol and cinnamon powder. The above-listed componentsmay be in the form of a salt or solvate thereof.

The oral preparation is produced by mixing the compound of the presentinvention with a filler, and if necessary, a binder, disintegrant,lubricant, coloring agent, flavoring agent, etc. and formulating themixture according to conventional procedures into, for example, apowder, subtle granules, granules, tablet, coated tablet, capsules orthe like. Resultant tablets and granules can be appropriately coatedwith, for example, sugar according to necessity. The syrups andinjection preparations can be prepared according to conventionalprocedures by adding a pH adjusting agent, solubilizer, and isotonizingagent, and if necessary, a dissolution aid, stabilizer, etc. Theexternal preparations can be produced according to conventionalprocedures not specifically limited. Base materials which may be used inthe present invention include various raw materials conventionally usedin pharmaceutical preparations, quasi drugs and cosmetics. Such rawmaterials include, for example, animal and vegetable oils, mineral oils,ester oils, waxes, higher alcohols, fatty acids, silicone oils,surfactants, phospholipids, alcohols, polyhydric alcohols, water-solublepolymers, clay minerals and purified water. If necessary, pH adjustingagents, antioxidants, chelating agents, antiseptics and antimolds,coloring agents, flavors, or the like can be added. In addition,components such as blood-flow accelerators, bactericides,anti-inflammatory agents, cell activators, vitamins, amino acids,humectants, keratolytic agents or the like be added according tonecessity. The ratio of the active ingredient to carriers may vary from1 to 90% by weight. When the compounds used in the present invention,the peptides used in the present invention or the polynucleotides usedin the present invention am used in the above-described treatment, it ispreferable to use those compounds, peptides or polynucleotides purifiedto 90% or more, preferably 95% or more, more preferably 98% or more,still more preferably 99% or more.

The effective dose of the pharmaceutical composition comprising thecompound of the present invention varies depending on the severity ofsymptom, the age, sex and body weight of the patient, administrationmode, type of the salt, specific type of the disease and other factors.Generally, the pharmaceutical composition may be administered to anadult (body weight: 60 kg) in one to several divided doses at a dailydose of about 30 μg to about 10 g, preferably 100 μg to 5 g, and morepreferably 100 μg to 100 mg for oral administration, or at a daily doseof about 30 μg to about 1 g, preferably 100 μg to 500 mg, and morepreferably 100 μg to 30 mg for injection administration. Consideringthat efficacy varies depending on the administration route, the requireddose is expected to vary widely. For example, it is expected that oraladministration requires a higher dose than intravenous injection. Whenadministered to children, the dose may be smaller than the dose foradults. These variations in the dose level can be adjusted by standardempirical optimization procedures which are well understood in theindustry.

The term “treatment” as used herein generally means a method forobtaining a desired pharmacological and/or physiological effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease and/or a symptom and may be therapeutic in terms ofpartially or completely curing a disease and/or an adverse effectattributed to the disease. The term “treatment” as used herein coversany treatment of a disease in a patient, preferably a human, andincludes at least one treatment selected from the following (a) to (c):

-   (a) preventing a disease or a symptom from occurring in a patient    who may be predisposed to the disease but has not yet been diagnosed    as having it;-   (b) inhibiting a disease symptom, i.e. preventing or delaying its    progress; or-   (c) relieving a disease symptom, i.e. causing regression or    elimination of the disease or symptom, or causing reversal of the    progress of the disease.

For example, as clinical symptoms of AD, progressive disorientation,memory loss and aphasia are enumerated. Finally, disablement, speechloss and akinesia occur. Pathological signs of AD includeneurofibrillary tangle, senile plaques and amyloid angiopathy. Toprevent the progress of AD is interpreted to mean to prevent the onsetor further progress of the clinical symptoms and/or pathological signsof AD. For example, in patients who do not have the clinical symptoms orpathological signs of AD, it is possible to prevent the progress ofclinical symptoms or pathological signs. In patients suffering from mildAD, it is possible to prevent the development of more severe AD forms.To delay the progress of AD is interpreted to mean to delay the point ofonset of AD-related symptoms and/or pathological signs, or to reduce thespeed of progress of AD that is determined by the speed of progress ofclinical symptoms and pathological signs. To reverse the progress of ADis interpreted to mean to relieve the severity of AD symptoms, i.e., tochange the severity of AD conditions of patients from severe to mild. Atthat time, the change to mild is indicated by decrease of clinicalsymptoms or pathological signs.

Diagnosis of AD in patients may be performed by various known methods.Typically, AD is diagnosed by combining clinical and pathologicalassessments. For example, the progress or severity of AD may be judgedusing Mini Mental State Examination (MMSE) (Mohs et al. (1996) IntPsychogeriatr 8: 195-203), Alzheimer's Disease AssessmentScale-Cognitive Subscale (ADCS-cog) (Galasko et al., (1997) AlzheimerDis Assoc Disord, 11 suppl 2: S33-9), Alzheimer's Disease CooperativeStudy-Activities of Daily Living (ADCS-ADL) (McKhann et al., (1984)Neurology 34: 939-944) and Criteria of National Institute of NeurologicCommunicative Disorders and Stroke-Alzheimer's Disease and RelatedDisorders Association (NINCDS-ADRDA) (Folstein et al., (1975) JPsychiatr Res 12: 189-198; McKhann et al., (1984) Neurology 34:939-944). Further, methods which evaluate various regions of the brainand enable the estimation of frequency of senile plaques orneurofibrillary tangle may be used (Braak et al., (1991) ActaNeuropathol 82: 239-259; Khachaturian (1985) Arch Neuro 42: 1097-1105;Mirra et al., (1991) Neurology 41: 479-486; and Mirra et al., (1993)Arch Pathol Lab Med 117: 132-144).

In another embodiment of the present invention, there is provided a kitfor γ-secretase assay, or a kit for identifying γ-secretase inhibitors,activators or modulators, each of which comprises EphA4 and, preferably,further comprises a substrate for γ-secretase other than EphA4(preferably APP and/or Notch). The kit of this embodiment may be used inthe screening method of the present invention.

The present invention provides a test kit for measuring the processingof EphA4 by γ-secretase. This kit of the present invention comprisesγ-secretase or a biological composition containing γ-secretase, and abiological composition containing EphA4. The kit may further comprise asubstrate for γ-secretase other than EphA4 (e.g., APP and/or Notch) or abiological composition containing such a substrate. Preferably, the kitcomprises a plurality of substrates for γ-secretase. It is preferredthat the kit comprises APP and/or Notch in addition to EphA4. Further,the kit comprises tools used in immunoblotting or Western blotting(e.g., reaction vessels, blotting membranes, etc.), reagents (e.g.,buffers, culture broths, anti-EphA4 antibodies, etc.), instructions, andso on. With the assay kit of the present invention, it is possible toevaluate whether or not a candidate compound affects the processing ofEphA4 by γ-secretase.

The present invention also includes the use of the above-described kitin measuring the processing of EphA4, or in the method of screening ortesting γ-secretase inhibitors.

EXAMPLES

Herein below, the present invention will be described in more detailwith reference to the following Examples and Preparation Examples.However, the present invention is not limited to these Examples, whichare provided only for the purpose of full disclosure of the presentinvention to those skilled in the art. It is not meant or even impliedthat the experiments described herein are all or only one experimentactually carried out. Although efforts have been made to guarantee theaccuracy of the numerical values used herein (e.g., volume, temperature,concentration, etc.), experimental errors and deviations are consideredto some extent. Thus, such values may be changed within a range whichdoes not depart from the scope of the present invention.

Example 1 Analysis of EphA4 Processing in EphA4-Transfected 294/EBNA-1Cell Strain

Whether or not EphA4 is a substrate for γ-secretase was evaluated usingHEK293 cells expressing EphA4 with an HA tag added to its C-terminus, inthe presence of a γ-secretase inhibitor. As the γ-secretase inhibitor,(2S)-2-{[(3,5-difluorophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide(hereinafter, sometimes referred to as “Compound E”) (AlexisBiochemicals) was used.

1. Experimental Conditions and Methods

(1) Cloning of Rat EphA4

RNA was purified from rat brain with Trisol (Invitrogen), followed bysynthesis of 1st strand cDNA with RNA PCR Kit (TaKaRa). Using 1 μl ofthe finally synthesized 1st strand cDNA product, rat EphA4 was amplifiedwith the following primers and Pfu (Stratagene):

primer1 XhoI site added: GAGCTCGAGGCCACCATGGCTGGGATTTTCTATTTCATC (SEQ IDNO: 89)

primer2 Nod site added: GAGGCGGCCGCGACAGGAACCATCCTGCCATGCATC (SEQ ID NO:90)

PCR conditions were as follows: first reaction at 95° C. for 2 minutes;then 35 cycles of (at 95° C. for 45 seconds→at 60° C. for 45 seconds→at72° C. for 6 minutes); then final reaction at 72° C. for 10 minutes. ThePCR product was purified with Quiaquick PCR purification kit (QIAGEN,treated with restriction enzymes XhoI (TaKaRa) and NotI (TaKaRa) andthen cloned into pBluescript (Stratagene).(2) Construction of a Gene Encoding Rat EphA4 with HA Tag Added to itsC-Terminus and an Expression Vector

pcDNA (Invitrogen) was treated with restriction enzymes XhoI (TaKaRa)and Nod (TaKaRa). EphA4 obtained by treating the pBluescript obtained in(1) above with restriction enzymes XhoI (TaKaRa) and Nod (TaKaRa) wasinserted into the resultant pcDNA to thereby prepare an expressionvector. This expression vector was constructed so that an HA tag isadded to the C-terminus of the incorporated EphA4. The DNA sequence ofthe resultant EphA4-HA was analyzed with a DNA sequencer (AppliedBiosystems Model 3130x1). The resultant DNA sequence is shown in SEQ IDNO: 3. In the resultant DNA sequence, the nucleotide at position 861 waschanged from c to t, compared with rat EphA4 (XM_(—)244186.4).(Hereinafter, this mutation is expressed as “c→t”. Other mutations willalso be expressed in the same manner.) Besides, the resultant DNAsequence had a→t mutation at position 1110, a→g mutation at position1278, a→g mutation at position 1320, c→t mutation at position 1623, c→tmutation at position 1626, c→t mutation at position 2208 and c→tmutation at position 2265, compared with rat EphA4 (XM_(—)244186.4).However, it was confirmed that they were 100% identical at the aminoacid level. This expression vector was prepared in large quantity withEndofree Plasmid Maxi Kit (QIAGEN).

The sequence spanning from g at position 2968 to t at position 2997 inthe DNA sequence of EphA4-HA (SEQ ID NO: 3) is a nucleotide sequenceencoding the HA tag.

The amino acid sequence of EphA4-HA is shown in SEQ ID NO: 4.

(3) Preparation of Cells Expressing a Gene Encoding Rat EphA4 with HATag Added to its C-Terminus

293/EBNA-1 cell strain (Invitrogen) was cultured in 10% FBS(Hyclone)/DMEM (Invitrogen) under 5% CO₂ at 37° C., followed bytransfection thereinto of a gene encoding rat EphA4 with an HA tag addedto its C-terminus with Lipofectamine 2000 (Invitrogen). After one dayculture under the same conditions, Compound E (γ-secretase inhibitor;Alexis Biochemicals) was added to the medium (final concentration: 50nM). Cells were cultured for another day under the same conditions.Then, the transfected HEK293 cells were collected with PBS (Sigma) andsonicated with a sonicator (Taitec VP-5S) to disrupt cells. Then, thequantity of protein was determined with Protein Assay Kit (BioRad).Samples (2 μg each) were taken from proteins obtained from CompoundE-added cells and Compound E-not added cells, respectively, andsubjected to SDS-PAGE, followed by Western blotting with an anti-HAantibody (Roche) (final concentration: 0.2 μg/ml).

2. Experimental Results

FIG. 1 shows the results.

In FIG. 1, the left lane represents the sample untreated with Compound Eand the right lane represents the sample treated with Compound E. “Full”represents the full-length of EphA4. “CTF” (C-terminal fragment)represents a region spreading from the transmembrane domain of EphA4(amino acid residue at position 547) to its C-terminal site; that is,this fragment is EphA4 undegraded product. When Compound E (γ-secretaseinhibitor) was added, a band around 50 kDa (CTF) was accumulatedspecifically. Since this band is equal in size to the region spreadingfrom the transmembrane domain of EphA4 to its C-terminal site, it hasbecome clear that EphA4 is cleaved by γ-secretase in HEK293 cells.

Example 2 Analysis of EphA4 Processing in Primary Culture of RatHippocampal Neurons

Whether or not EphA4 is a substrate for γ-secretase was evaluated usinga primary culture of rat hippocampal neurons in the presence of aγ-secretase inhibitor. As the γ-secretase inhibitor,(2S)-2-{[(3,5-difluorophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide(Compound E) (Alexis Biochemicals) was used.

1. Experimental Conditions and Methods

(1) Preparation of Primary Culture of Neurons

The hippocampus was isolated from embryonic day 18 SD rats (CharlesRiver) and subjected to culture. Specifically, embryos were removedaseptically from pregnant rats under etherization. The brain was removedfrom each embryo and dipped in 20% FBS (Hyclone)/HBSS (Sigma). Thehippocampus was collected from the thus obtained brain under astereomicroscope. The collected hippocampus was treated in an enzymesolution containing 0.25% trypsin (Invitrogen) and 0.5 mg/ml DNase(Sigma) at 37° C. for 10 minutes to disperse cells. This enzyme reactionwas terminated by adding 20% FBS (Hyclone)/HBSS (Sigma). Then, 2 ml ofMSS (Sigma) was added to the resultant cells. The HBSS-added cell masswas re-dispersed by gentle pipetting. The resultant neuron suspensionwas diluted with a medium and plated in 10 cm dishes at an initial celldensity of 1.5×10⁶ cells/dish. As the medium, MEM medium (Invitrogen)supplemented with 10% FBS (Hyclone), 1× B27 Supplement (Invitrogen, 2 mML-glutamine (Invitrogen) and 25 μg/ml insulin (Sigma) was used. Theplated cells were cultured in an incubator under 5% CO₂ and 95% air at37° C. for 3 days. Then, a half volume of the medium was exchanged witha cytosine β-D-arabinofuranoside hydrochloride (AraC)-containing mediumin order to inhibit the growth of glial cells. As the AraC-containingmedium, MEM medium (Invitrogen) supplemented with 5% FBS (Hyclone), 1×B27 Supplement (Invitrogen), 0.5 mM L-glutamine (Invitrogen), 25 μg/mlinsulin (Sigma) and 8 μM AraC (Sigma) was used. Two weeks after thestart of culture, Compound E (final concentration: 50 nM) was added, andthe cells were cultured for another week. Then, cells were collectedwith PBS and subjected to quantitative determination of protein. Proteinsamples (10 μg each) were subjected to SDS-PAGE and Western blottingwith an anti-EphA4 antibody (Upstate) (1/500 dilution).

2. Experimental Results

FIG. 2 shows the results.

In FIG. 2, the left lane represents the sample untreated with Compound Eand the right lane represents the sample treated with Compound E. “Full”represents the full-length of EphA4. “CTF” represents a region spanningfrom the transmembrane domain of EphA4 to its C-terminal site. WhenCompound E (γ-secretase inhibitor) was added, a band around 50 kDa (CTF)was accumulated specifically. Since this band is equal in size to theregion spreading from the transmembrane domain of EphA4 to itsC-terminal site, it has become clear that EphA4 is cleaved byγ-secretase in hippocampal neurons.

The technical terms used herein are used only for the purpose ofillustrating a specific embodiment and not intended to limit theembodiment.

Unless otherwise specifically defined, all technical terms andscientific terms used herein have the same meaning as generallyunderstood by those skilled in the art. Although any methods andmaterials similar to or equivalent to those described herein may be usedin the practice or test of the present invention, those skilled in theart can consult the description provided herein, for preferable methodsand materials.

All publications cited herein are incorporated herein by reference intheir entirety for the purpose of describing and disclosing, forexample, the cell lines, constructs and methods described in thepublications that are used in connection with the present invention, orincorporated herein as references with respect to the disclosure of thecompound identification method, screening method and methodologiestherefor, and composition of the present invention; such publicationsmay be used for the practice of the present invention.

Sequence Listing Free Text

SEQ ID NO: 89: synthetic DNA

SEQ ID NO: 90: synthetic DNA

What is claimed is:
 1. A kit for performing a γ-secretase assay, comprising EphA4.
 2. A test kit for measuring the processing of EphA4 by γ-secretase, comprising a first biological composition containing γ-secretase or a biologically active fragment thereof and a second biological composition containing EphA4.
 3. The kit according to claim 1, further comprising γ-secretase. 